CN108271781A - It is a kind of from the parahydroxyben-zaldehyde and its production method of pseudomonad and application - Google Patents
It is a kind of from the parahydroxyben-zaldehyde and its production method of pseudomonad and application Download PDFInfo
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- CN108271781A CN108271781A CN201810019917.4A CN201810019917A CN108271781A CN 108271781 A CN108271781 A CN 108271781A CN 201810019917 A CN201810019917 A CN 201810019917A CN 108271781 A CN108271781 A CN 108271781A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
- A01N35/04—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing aldehyde or keto groups, or thio analogues thereof, directly attached to an aromatic ring system, e.g. acetophenone; Derivatives thereof, e.g. acetals
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Abstract
The invention discloses a kind of parahydroxyben-zaldehyde from pseudomonad and its production method and applications.The present invention provides the application of parahydroxyben-zaldehyde or derivatives thereof or its pharmaceutically acceptable salt in preventing the plant disease caused by plant pathogenic fungi, or the application in the pesticide or biological prevention and control agent for preparing prevention plant disease caused by plant pathogenic fungi.The present invention passes through Antibacterial Activity, demonstrate parahydroxyben-zaldehyde has good inhibitory activity to plant pathogenic fungi, it can inhibit the growth of sugarcane whip smut, to effectively prevent smut of sugarcane, reference is provided for prevention and control plant disease caused by plant pathogenic fungi.
Description
Technical field
The invention belongs to technical field of biological control.More particularly, to a kind of para hydroxybenzene from pseudomonad
Formaldehyde and its production method and application.
Background technology
Parahydroxyben-zaldehyde is important the intermediate of medicine, fragrance, pesticide, in medical industry, main synthesis hydroxyl
Ampicillin (Amoxicillin), Trimethoprim first trimethoprim (TMP), 3,4,5- trimethoxies formaldehyde, to hydroxyl
Phthalic anhydride propylhomoserin, amoxycillin cephalosporin, artificial Rhizoma Gastrodiae, farrerol, esmolol etc.；For synthesizing chinese cymbidium in perfume industry
The fragrance such as element, Ethyl vanillin, heliotropin, syringaldehyde, anisaldehyde and raspberry ketone；Herbicide bromine is mainly used as in pesticide
The synthesis of cyanophenyl and chloroxynil；Be mainly used in chemical industry synthesize P-hydroxybenzoic acid, to hydroxy carboxylic acid benzyl ester, acetic acid to hydroxyl
Base phenol ester etc..
Smut of sugarcane (Sugarcane smut) is invaded by sugarcane whip smut (Sporisorium scitamineum)
Fungal disease caused by dye is one of the worldwide important disease for influencing sugarcane economy and production safety, seriously restricts sweet
The sound development of sugarcane industry.China is cane planting big country, be mainly distributed on Guangdong, Guangxi, Yunnan, Hainan, Fujian, Sichuan and
The areas such as Taiwan.Due to cultivar unification, with generation and popular, the sugarcane whip smut biological strain of smut of sugarcane
Constantly change so that there is disease report in China cane planting region, and it is further serious to fall ill, and seriously affects China's sugarcane production
The development of industry, every year caused by smut of sugarcane direct economic loss up to 5,000,000,000 yuan.The disease difficulty of prevention and cure is big, and there has been no direct
Act on the chemical agent in field.
In recent years, there is report of the part about smut of sugarcane biological control, but large area long term repeatability in recent years
Ground uses the identical fungicide of mechanism of action, it is easy to so that smut of sugarcane is developed immunity to drugs, decline so as to cause preventive effect, even
It is completely ineffective, great economic loss is caused to sugarcane production.Therefore, the screening and development of new medicament have great importance,
The fungicide using different role mechanism should be replaced in production, to delay the appearance of pathogen drug resistance.
By literature search, the open report with parahydroxyben-zaldehyde solution prevention smut of sugarcane is not found.
Invention content
The technical problem to be solved by the present invention is to overcome above-mentioned the deficiencies in the prior art, provide a kind of from pseudomonad
Parahydroxyben-zaldehyde and its production method and parahydroxyben-zaldehyde or derivatives thereof or its pharmaceutically acceptable salt anti-
Control the application in the plant disease caused by plant pathogenic fungi.
The object of the present invention is to provide parahydroxyben-zaldehyde or derivatives thereof or its pharmaceutically acceptable salt prevention by
Application in plant disease caused by plant pathogenic fungi.
It is a further object of the present invention to provide a kind of fungicide of plant pathogenic fungi.
Another object of the present invention is to provide a kind of production method of parahydroxyben-zaldehyde.
Another object of the present invention be to provide the fermentation broth extract from pseudomonad prevent it is true by pathogenic
Application in microbial plant disease.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
The present invention relates to parahydroxyben-zaldehyde or derivatives thereof or its pharmaceutically acceptable salt in prevention by pathogenic
Application in fungus-caused plant disease, or in pesticide or the life for preparing prevention plant disease caused by plant pathogenic fungi
Application in anti-preparation.
The present invention passes through Antibacterial Activity, it was demonstrated that parahydroxyben-zaldehyde has good inhibition to plant pathogenic fungi
Activity.Parahydroxyben-zaldehyde is suitable for the requirement of plant disease chemical prevention as fungicide, efficient, low toxicity.
Preferably, the plant pathogenic fungi is smut；The plant disease is smut of sugarcane, the black powder of corn tumor
Disease or other through heterothallic fungal disease such as phytophthora root rot, peronospora tabacina, rice blast, rust etc..
It is highly preferred that the smut is sugarcane whip smut, corn tumor smut, Tilletia indica, wheat
Net bunt powder bacterium or ustilaginaceae etc..Experiment finds that parahydroxyben-zaldehyde is to sugarcane whip smut, corn tumor smut etc.
A variety of smut have excellent bacteriostatic activity.
Preferably, it is applied to inhibit the growth of plant pathogenic fungi.
Preferably, the plant is sugarcane, corn, sorghum, rice, wheat, peanut or lichee etc..
Preferably, parahydroxyben-zaldehyde inhibits the minimum of plant pathogenic fungi growth to use a concentration of 3mM.
When a concentration of 2mM of parahydroxyben-zaldehyde, the mycelia growth of sugarcane whip smut is weaker than blank control group, cannot be outside
Prolong growth mycelia；And when parahydroxyben-zaldehyde concentration is higher than 3mM, the growth of sugarcane whip smut is completely inhibited, explanation
The parahydroxyben-zaldehyde of a concentration of 3mM has significant inhibiting effect in sweet black whip smut growth period.
The present invention also provides a kind of bacteriostatic agent of plant pathogenic fungi, containing parahydroxyben-zaldehyde or derivatives thereof or its
Pharmaceutically acceptable salt.
Preferably, the plant pathogenic fungi is smut；The smut is sugarcane whip smut, the black powder of corn tumor
Bacterium, Tilletia indica, wheat net bunt powder bacterium or ustilaginaceae etc..
The present invention also provides a kind of production methods of parahydroxyben-zaldehyde, mainly include the following steps that：
S1. solid fermentating mode is used, pseudomonad is inoculated in PDA culture medium, 36~60h is cultivated at 26~30 DEG C
After wipe bacterium colony, obtain fermentation medium；
S2. be soaked in after above-mentioned fermentation medium being cut into small pieces 2~3 times of volumes by ethyl acetate, methanol and ice vinegar
In the mixed solution of acid composition, supernatant is collected by filtration, the organic solvent in supernatant is removed after reduced pressure, with 2~3 times of bodies
Long-pending ethyl acetate aqueous phase extracted solution 2~3 times, obtains fermentation broth extract；
S3. fermentation broth extract obtained by S2 is obtained into parahydroxyben-zaldehyde through chromatography；The chromatographic separating process packet
Include silica gel column chromatography, medium pressure liquid chromatography separates, analyzes type high performance liquid chromatography separation or semipreparative high performance liquid chromatography point
From.
This method is easy to operate quickly, and the compound of opposed polarity is efficiently separated according to the polarity of organic solvent, can be carried
The separative efficiency of high compound.
Preferably, the pseudomonad is pseudomonad ST4, which is preserved in Chinese allusion quotation on 14th in September in 2015
Type culture collection, deposit number CCTCCNO：M2015526, preservation address are Wuhan, China.
Preferably, the formula (1L) of PDA culture medium is in step S1：200g peeled potatoes are cut after fritter boils 30min
20g glucose and 18g agar powders is added in filtering, filtrate.
Preferably, ethyl acetate in step S2：Methanol：The volume ratio of glacial acetic acid is 70~90：10~20：4~6.
It is highly preferred that ethyl acetate in step S2：Methanol：The volume ratio of glacial acetic acid is 80：15：5.
Particularly preferably, the production method of the parahydroxyben-zaldehyde, mainly includes the following steps that：
S1. the pseudomonad ST4 bacterium solutions being incubated overnight are inoculated in PDA culture medium, sterile gauze is used after 28 DEG C of culture 48h
Bacterium colony is wiped, fermentation medium is obtained；
S2. be soaked in after fermentation medium being cut into small pieces 3 times of volumes by ethyl acetate：Methanol：Glacial acetic acid presses 80：
15：In the mixed solution of 5 volume ratio composition, supernatant is collected by filtration after shaking 2h；The second in removal supernatant is concentrated under reduced pressure
The organic solvents such as acetoacetic ester, methanol, stay aqueous phase solution；With the ethyl acetate aqueous phase extracted solution 3 times of 3 times of volumes, collect respectively
Water phase and organic phase, obtain fermentation broth extract；
S3. by fermentation broth extract obtained by S2 through the separation of ClaricepFlashSilica (CS) standard silica gel column chromatography, cloth
Parahydroxyben-zaldehyde is partly prepared in the separation of pressure separator, high performance liquid chromatography separation and HPLC in strange.
Preferably, it is (1L) that LB liquid medium, culture medium prescription are used when being incubated overnight in step S1：Yeast extract
5g, tryptone 10g, sodium chloride 10g.
Preferably, the standard silicagel columns of ClaricepFlashSilica (CS) described in step S3 are that Agela biotechnologys are public
The positive silicon 120g containing 60 mesh of production is taken charge of, can tolerate pressure is 180psi；60~100 mesh of stationary phase silica gel, mobile phase be with
Different proportion (100:0、99:1、98:2、95:5、9:1、8:2、1:100) mixed liquor of the chloroform and methanol mixed.
Preferably, the semipreparative methods of HPLC described in step S3, mainly include the following steps：
S11. sample to be prepared being dissolved in methanol, a concentration of 50~100mg/mL is loaded on sample bottle after impurity screening, into
Sample amount is 50~100 μ L, and eluent is the mixed liquor of acetonitrile and ultra-pure water, eluent flow rate 3mL/min；Wherein HPLC half makes
Standby elution process is：0~30min, the concentration of acetonitrile is by 5%~100% gradient elution chromatography column；30~35min, 100%
Acetonitrile rinses chromatographic column；35~40min, 5% acetonitrile balance chromatographic column；
S12. mixed liquor is collected in batches according to preparation time and chromatogram effect, each component is evaporated, is weighed, is dissolved in suitable
HPLC and TLC Analyze ＆ separate effects are used after the methanol of amount respectively, while detecting the bacteriostatic activity of each component；
Wherein, HPLC and TLC then are evaporated, title analysis shows that for single substance and the component with bacteriostatic activity
It is dissolved separately in methanol and deuterated methanol solution after weight, is analyzed for LC-MS and nuclear magnetic resonance chromatography；To active, but
The more component of HPLC and TLC analysis substances, carries out HPLC and prepares again, until single for component.
The present invention active material single to component carries out further nuclear magnetic resonance (NMR) and mass spectrum (MS) analysis, tool
Body method is as follows：
S21：It is molten with suitable deuterated methanol after the component of only one point is evaporated on and TLC plates single to HPLC chromatogram peak
Solution is loaded on nuclear magnetic tube, carries out nmr analysis after sealing, then determine that separate substance can according to nuclear magnetic resonance spectroscopy result
Structure existing for energy determines molecular structure in conjunction with mass spectral analysis；Wherein, the analysis method includes nuclear magnetic resonance spectroscopy 1HNMR,
Carbon-13 nmr spectra 13CNMR, nuclear-magnetism carbon compose dept135 spectrums, dept90 spectrums, two-dimentional nuclear spectrum (COSY, QC, BC)；
S22：After substance after isolating and purifying is dissolved in proper amount of methanol, with mass spectrograph detect separate substance molecular weight and can
The chemical constitution of energy, the molecular weight of separate substance is determined according to mass spectrometry results, substance is determined in conjunction with nmr spectrum
Molecular structure.
Preferably, 1HNMR and 13CNMR described in step S21 is divided using BrukerAvanceIII600 at 600MHz
Analysis.
Preferably, MS analyzers described in step S22 are BrukermaXisimpact.
The present invention also provides a kind of fermentation broth extracts of pseudomonad ST4, containing parahydroxyben-zaldehyde, by above-mentioned
Step S1, step S2 are obtained in production method.
In addition, application of the above-mentioned fermentation broth extract in preventing the plant disease caused by plant pathogenic fungi, or
Application in the pesticide or biological prevention and control agent for preparing prevention plant disease caused by plant pathogenic fungi, also in the guarantor of the present invention
Within the scope of shield.
Compared with prior art, the present invention has the advantages that：
1, parahydroxyben-zaldehyde of the present invention or derivatives thereof or its pharmaceutically acceptable salt are being prevented by plant
There is important use, it is particularly possible to inhibit the growth of sugarcane whip smut, to effectively in plant disease caused by disease fungus
Smut of sugarcane is prevented, green safe pesticide is provided for prevention and control plant disease caused by plant pathogenic fungi.
2, parahydroxyben-zaldehyde is a kind of pollution-free and environment amenable micromolecular compound, and it is to non-target life
Object and safety of human and livestock, pathogen are poor to its drug resistance, can ensure the high-quality of agricultural product, meet the requirement of sustainable development,
It is studied and market application prospect is wide.
3, the present invention provides a kind of methods that solid fermentation extracts the effective metabolite of biocontrol microorganisms, to obtain effective active
The lower biocontrol microorganisms of physical efficiency provide good reference.
Description of the drawings
Fig. 1 is the TLC and bacteriostatic activity analysis of 4 fractions (ST4-1~ST4-4) of bacterial strain ST4 metabolins.A is metabolized for ST4
The TLC of 4 fraction ST4-1~ST4-4 of object is analyzed；It is dissolved in methanol per component, final concentration of 50mg/mL respectively takes 0.5 μ L points to exist
Silica gel plate is placed in containing a small amount of solvent (chloroform by TLC tablets：Methanol=9：1) staining jar, solvent are slightly below point sample position
It sets, ascending method expansion, observing band under ultraviolet lamp after expansion detaches situation, then invades bubble and high temperature with 10% sulfuric acid ethyl alcohol and show
Color；The bacteriostatic activity that B is 4 fraction ST4-1~ST4-4 of ST4 metabolins is analyzed；MAT-1 the and MAT-2 mixed liquors of 0.5 μ L
(OD600≈ 1.5) point is on PDA agar strips, and the aseptic filter paper for having soaked fraction is put in one end, and tablet is seen two days later in 28 DEG C of cultures
Examine mycelia upgrowth situation.
Fig. 2 is HPLC, the TLC and bacteriostatic activity for 9 component ST4-2-1~ST4-2-9 that active component ST4-2 is isolated
Analysis.A is the HPLC collection of illustrative plates of 9 component ST4-2-1~ST4-2-9 of ST4-2, and UV absorption is λ=254nm.B is ST4-2
9 component ST4-2-1~ST4-2-9 TLC analysis.C is the antibacterial work of 9 component ST4-2-1~ST4-2-9 of ST4-2
Property analysis.
Fig. 3 is the HPLC collection of illustrative plates of single-activity component ST4-2-6 and standard sample parahydroxyben-zaldehyde.
Fig. 4 is fungistatic effect of the compound parahydroxyben-zaldehyde to sugarcane whip smut.
Specific implementation mode
It is further illustrated the present invention below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are purchased in market.
The extraction of 1 pseudomonad ST4 metabolins of embodiment and its detection of bioactivity
1, the extraction of bacterial strain ST4 metabolins
(1) the ST4 bacterium solutions being incubated overnight are inoculated on PDA plate (13 × 13cm), with sterilizing yarn after 28 DEG C of culture 48h
Cloth wipes bacterium colony, obtains fermentation medium；
(2) be soaked in after fermentation medium being cut into small pieces 3 times of volumes by ethyl acetate：Methanol：Glacial acetic acid presses 80：
15：In 5 volume ratio composition mixed solution, supernatant is collected by filtration after shaking 2h；With Rotary Evaporators by the second in supernatant
The organic solvents such as acetoacetic ester, methanol remove, and stay aqueous phase solution；
(3) with the ethyl acetate aqueous phase extracted solution of 3 times of volumes 3 times, water phase and organic phase are collected respectively, organic phase is made
It is evaporated with Rotary Evaporators, thickening temperature is 45 DEG C, after weighing weight, a small amount of acetic acid ethyl ester extract is taken to be dissolved in suitable first
In alcohol, for detecting its bacteriostatic activity；Water phase then with the extracting n-butyl alcohol of 3 times of volumes 3 times, collects water phase and n-butanol respectively
Phase, n-butanol mutually rotates be evaporated after weigh weight, suitable methanol is dissolved in, for detecting n-butyl alcohol extract to the black powder of sugarcane whip
The inhibiting effect of bacterium.
2, the detection of active material：
(1) prepare 0.6 × 0.6cm (or 0.6cm diameters) size aseptic filter paper piece, by PDA plate be cut into 0.8cm wide,
The band of 6cm long, one end, which places aseptic filter paper piece and draws 100 μ L, detects liquid, after filter paper dries, by MAT-1 and MAT-2
The uniform place of mixed bacteria liquid to PDA items take, and after 28 DEG C of cultures generate white fluffy mycelia to blank control group, record
Inhibition situation of the different testing sample to sugarcane smut.
(2) it is to obtain enough crude extracts to detach for consequent activities substance, the present invention obtains 20L biocontrol microorganisms ST4 altogether
Culture.Ethyl acetate extraction obtains 38.82g runic objects, and extracting n-butyl alcohol obtains 12.51g crude extracts.Take a small amount of acetic acid second
Ester extract and n-butyl alcohol extract are dissolved in suitable methanol, are made into the solution of a concentration of 50mg/mL respectively, and detect it to sweet
The influence of sugarcane whip smut.
3, testing result
(1) bacteriostatic test as a result, it has been found that, in the filter paper containing n-butyl alcohol extract, the mycelia of sugarcane whip smut can also give birth to
It is long, but growing way is relatively weaker than space management；And the band containing acetic acid ethyl ester extract, the sugarcane whip smut close to filter paper protect
It holds as bacterium shape, white hypha cannot be formed, and lower closer to the colony density of filter paper.
(2) above-mentioned the experiment results show that in acetic acid ethyl ester extract contain certain inhibition sugarcane whip smut normal growths
Substance, and be free of in n-butanol extract or only minimal amount of active material.Therefore, by acetic acid ethyl ester extract (ST4)
Separation for consequent activities substance.
2 pseudomonad ST4 effective active matters of embodiment isolate and purify
1, isolation and purification method
By the acetic acid ethyl ester extract of 1 gained of embodiment successively through ClaricepFlashSilica (CS) standard silicagel column
The separation such as the very middle pressure separator separation of chromatography, cloth, half preparation of high performance liquid chromatography separation and HPLC, the specific method is as follows：
(1) standard silica gel column chromatography detaches：
1) loading：5~the 6g of acetic acid ethyl ester extract for accurately weighing pseudomonad ST4 every time is dissolved with suitable methanol,
2 times of 60~100 mesh silica gel are added, stir, after air-drying methanol, silica gel with sample is added into sample column,
Be connected with ClaricepFlashSilica (CS) standard silicagel column, at the same with cloth it is strange in pressure separator and analysis level methanol and
Chloroform is connected, and checks whether each junction is normal；
2) it detaches：It is washed after being mixed chloroform and methanol in the following proportions under 18psi pressure using the strange middle pressure separator of cloth
De- sample, eluent are followed successively by chloroform：Methanol=100：0、99：1、98：2、95：5、9：1、8：2 and 0：100, elution flow rate is
30mL/min, each gradient elution 10min, by per gradient elution 300mL eluent Fraction collections；
3) it collects：The eluent of collection is evaporated respectively with Rotary Evaporators, weighs weight, and by isolate with suitable
Methanol dissolves, and uses high performance liquid chromatography (HPLC) to analyze different eluents with thin layer chromatography (TLC) respectively and detaches situation.
(2) high performance liquid chromatography separation：High performance liquid chromatograph be Agilent 1260, ultraviolet detection wavelength be 202,210,
230,254 and 280nm, chromatographic column are C18 analytical columns and C18 semi-preparative columns, and mobile phase is acetonitrile and ultra-pure water.Analysis method
For：
1) sample for being dissolved in methanol of step (1) is settled to 10~50mg/mL, sample bottle, sample is loaded on after impurity screening
2 μ L of product sample introduction, eluent are the mixed liquor of acetonitrile and ultra-pure water, eluent flow rate 1mL/min；
2) HPLC elution requirements are 0~30min, and the concentration of acetonitrile is by 5%~100% gradient elution chromatography column；30~
35min, 100% acetonitrile rinse chromatographic column；35~40min, 5% acetonitrile balance chromatographic column；
3) chromatogram is observed, the separating effect of different component is analyzed.
(3) prepared by high performance liquid chromatography half：
1) sample to be prepared of step (2) is dissolved in methanol, a concentration of 50~100mg/mL is loaded on sample after impurity screening
Bottle, 50~100 μ L of sample feeding, eluent are the mixed liquor of acetonitrile and ultra-pure water, eluent flow rate 3mL/min.HPLC items
Part is 0~30min, and the concentration of acetonitrile is by 5%~100% gradient elution chromatography column；30~35min, 100% acetonitrile rinse chromatography
Column；35~40min, 5% acetonitrile balance chromatographic column；
2) mixed liquor is collected in batches according to preparation time and chromatogram effect, be evaporated each component with Rotary Evaporators,
It weighs, is dissolved in after suitable methanol and uses HPLC and TLC Analyze ＆ separate effects respectively, while detecting the bacteriostatic activity of each component；
3) it to HPLC and TLC analysis shows that be single substance, and is evaporated again with the component of bacteriostatic activity, after weighing respectively
It is dissolved in methanol and deuterated methanol is used for LC~MS and nuclear magnetic resonance chromatography；To active, but HPLC and TLC analysis substance compared with
More components carries out HPLC and prepares again, until component is single.
(4) TLC is analyzed：The TLC analyses of each section sample prepared through silica gel separation and HPLC, can intuitively observe institute
The purity of separation component.TLC silica gel plates are the aluminum silica gel plate of purchase, and solvent is chloroform and methanol (9：1) mixed liquor, display
The sulfuric acid ethyl alcohol that agent is 10%.Specific method is：
1) it is dense to prepare sample with methanol solution for the silica gel plate that aluminum silica gel plate is cut into suitable size by sample size
Degree is 10~50mg/mL, the point sample position of each sample of Pencil marks and sample ID, and a small amount of sample is drawn using capillary
Point is in corresponding position；
2) silica gel plate is placed in the staining jar containing a small amount of solvent, solvent is slightly below point sample position, ascending method expansion, exhibition
Band is observed under ultraviolet lamp after opening and detach situation, then invade bubble and high temperature colour developing with 10% sulfuric acid ethyl alcohol, by ultraviolet lamp and show
Show the material purity for observing different samples and separation situation.
2, result is isolated and purified
(1) merged according to TLC results and obtain 4 parts of crude extract component, respectively ST4-1,0.7328g of 0.0821g
The ST4-4 of the ST4-3 and 12.3515g of ST4-2,2.1634g, and TLC analyses and activity identification (figure are carried out to this 4 parts of isolates
1).From fig. 1, it can be seen that there may be a variety of substances for inhibiting sugarcane whip smut in the metabolin of ST4.TLC analysis shows, ST4-
2 bands are simple, and polarity is moderate, and ST4-3 polarity is larger and TLC bands are fuzzy, and it is more complicated that there are substances.Therefore, using ST4-2
Carry out half preparative separations of HPLC.
(2) HPLC of ST4-2 is analysis shows that (Fig. 2A), ST4-2 combinations ultraviolet absorption peak and disengaging time have collected 9 respectively
Part sample, respectively 0~8.6min of ST4-2-1 acquisition times, 74.4mg；ST4-2-2 8.6~9.4min of acquisition time,
18.6mg, ST4-2-3 acquisition time 9.4~10min, 35.7mg；ST4-2-4 acquisition times 10~11.5min, 6.6mg；ST4-
2-5 acquisition times 11.5~18.6min, 37mg；ST4-2-6 acquisition times 18.6~20min, 9.1mg；When ST4-2-7 is collected
Between 20~23.4min, 36.8mg；ST4-2-8 acquisition times 23.4~24.5min, 6.9mg；ST4-2-9 acquisition times 24.5~
40min, 307.5mg.
(3) TLC is analysis shows that (Fig. 2 B), and in the HPLC separation components of ST4-2, ST4-2-6 and ST4-2-8 are containing only an item
Band, purity is higher, is shown as the possibility containing only a compound, and other components are still mixture, and regularization condition is needed to continue point
From.Each separation component activity analysis shows (Fig. 2 C) that multiple separation components of ST4-2 have inhibiting effect to sugarcane whip smut,
Wherein ST4-2-4 and ST4-2-6 inhibits the growth of sugarcane whip smut obvious, the sugarcane whip smut bacterium colony growth of nearly filter paper
It is weaker in addition without growth sign；ST4-2-8 can also inhibit the growth of bacterium colony；Other parts to sugarcane whip smut inhibiting effect not
Obviously.
It follows that biocontrol microorganisms pseudomonad ST4 is various to the inhibiting effect of sugarcane whip smut, it is generated
Substance is not single but many kinds of substance immixture.This inhibits sugarcane whip smut to provide biocontrol microorganisms ST4
Many kinds of measures, is more advantageous to prevention of the field to smut of sugarcane.
The molecular formula and Structural Identification of 3 compound ST4-2-6 of embodiment
1, Structural Identification：ST4-2-6 is obtained to embodiment 2 and carries out NMR and LC-MS analyses, identifies its chemical constitution.
2, qualification result：
The NMR of ST4-2-6 analysis shows, which includes a benzene ring structure, and No. 1 position of phenyl ring contains that there are one hydroxyls
Base, there are one aldehyde radicals for No. 4 positions, may be parahydroxyben-zaldehyde；LC-MS analysis shows, the molecular weight of the substance is 122.12, with
Parahydroxyben-zaldehyde is consistent；HPLC analysis shows the retention time of (Fig. 3), ST4-2-6 and parahydroxyben-zaldehyde is 5.9min,
And the peak type and three-dimensional structure of the two are completely the same, therefore, ST4-2-6 are accredited as parahydroxyben-zaldehyde.
Inhibition of 4 parahydroxyben-zaldehyde of embodiment to sugarcane whip smut
1, test drug
Pass through the separation to ST4 metabolites, it is determined that the structure of compound ST4-2-6.Activity analysis discovery, ST4-2-
6 (parahydroxyben-zaldehydes) have the potentiality for inhibiting the growth of sugarcane whip smut.From the limited public affairs of Shanghai Aladdin biochemical technology share
Department's purchase sterling, to verify inhibition of the compound to sugarcane whip smut.
2, fungistatic effect
As shown in Figure 4, the results showed that, when a concentration of 2mM of parahydroxyben-zaldehyde, the mycelia growth of sugarcane whip smut is weak
It, cannot be to extension growth mycelia in control group；And when its concentration is higher than 3mM, then completely inhibit sugarcane whip smut
Growth.Illustrate that parahydroxyben-zaldehyde has significant bacteriostasis to sugarcane whip smut.
Claims (10)
1. parahydroxyben-zaldehyde or derivatives thereof or its pharmaceutically acceptable salt are planted in prevention caused by plant pathogenic fungi
Application in object disease, or answering in the pesticide or biological prevention and control agent for preparing prevention plant disease caused by plant pathogenic fungi
With.
2. application according to claim 1, which is characterized in that the plant pathogenic fungi is smut.
3. application according to claim 1, which is characterized in that the plant disease is smut of sugarcane, corn tumor is black
Powder disease, phytophthora root rot, peronospora tabacina, rice blast or rust.
4. application according to claim 2, which is characterized in that the smut is sugarcane whip smut, corn tumor is black
Powder bacterium, Tilletia indica, wheat net bunt powder bacterium or ustilaginaceae.
5. a kind of bacteriostatic agent of plant pathogenic fungi, which is characterized in that contain parahydroxyben-zaldehyde or derivatives thereof or its pharmacy
Upper acceptable salt.
6. a kind of production method of parahydroxyben-zaldehyde, which is characterized in that include the following steps：
S1. solid fermentating mode is used, pseudomonad is inoculated on PDA plate, is wiped after cultivating 36~60 h at 26~30 DEG C
Bacterium colony is removed, fermentation medium is obtained；
S2. be soaked in after above-mentioned fermentation medium being cut into small pieces 2~3 times of volumes by ethyl acetate, methanol and glacial acetic acid group
At mixed solution in, supernatant is collected by filtration, the organic solvent in supernatant is removed after reduced pressure, with 2~3 times of volumes
Ethyl acetate aqueous phase extracted solution 2~3 times, obtains fermentation broth extract；
S3. fermentation broth extract obtained by S2 is obtained into parahydroxyben-zaldehyde through chromatography.
7. production method according to claim 6, which is characterized in that the pseudomonad is pseudomonad ST4, the bacterium
Strain is preserved in China typical culture collection center on the 14th in September in 2015, and deposit number is CCTCC NO：M2015526.
8. production method according to claim 6, which is characterized in that ethyl acetate in step S2：Methanol：The body of glacial acetic acid
Product is than being 70~90：10~20：4~6.
9. a kind of fermentation broth extract of pseudomonad ST4, which is characterized in that contain parahydroxyben-zaldehyde.
10. application of the fermentation broth extract described in claim 9 in preventing the plant disease caused by plant pathogenic fungi, or
Application in the pesticide or biological prevention and control agent for preparing prevention plant disease caused by plant pathogenic fungi.
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